AGR2 and TFF3 Regulation in the Diagnosis and Treatment of Cancer

ABSTRACT

A method for assessing tumor progression is described by assessing AGR2 and/or TFF3 expression in a biological sample after induction of a physiological stress, such as hypoxia or serum deprivation in an enriched sample. Assessing the role of these indicators and their expression levels in an enriched CTC sample provides diagnostic and prognositic information on a patient. This method is also useful as a pharmatool in drug discovery.

BACKGROUND OF THE INVENTION

1. Field of the Invention

This invention relates generally to gene specific amplification,analysis and profiling of cytosolic biomolecules useful in the fields ofoncology, diagnostic testing and pharmacogenomics (personalizedmedicine). The invention is particularly useful in such fields as cancerscreening, selecting (identification and stratification of therapyresponders/non-responders) and monitoring for chemotherapy treatment, orcancer recurrence.

2. Description of Related Art

The ability of tumor cells to metastasize to distant organs isresponsible for most cancer deaths. In recent years, several studieshave used gene expression profiling analysis on primary and metastatictumors resulting in identification of genes having potential roles intumor progression towards metastasis. However, despite recent advances,the molecular signaling mechanisms associated with metastasis remainpoorly understood. Detection and characterization of disseminated tumorcells is beginning to aid in the dissection of the metastatic cascade,or the different events that lead to primary and secondary metastases inpatients with cancer. In fact, enumeration of circulating tumor cells(CTCs) has recently been shown to be an independent predictor ofprogression-free survival and overall survival in patients withmetastatic breast cancer (Cristofanilli, M., et al., 2004. CirculatingTumor Cells, Disease Progression, and Survival in Metastatic BreastCancer. N. Eng. Jour Med 351, 781-791).

In addition to enumeration, many studies have been published regardinggene expression profiling of CTCs. These studies have yielded usefulclinical information, but are limited to the evaluation of genespreviously identified in solid tumors (O'Hara S M, et al., 2004.Multigene Reverse Transcription-PCR Profiling of Circulating Tumor Cellsin Hormone-Refractory Prostate Cancer. Clin Chem. 50, 826-35.).Characterization of novel signaling mechanisms required for expressionof genes prevalently expressed in CTCs will be crucial to gaininginsight into the multi-step processes of tumor progression towardsmetastasis and should aid in the design of more targeted therapies.

A major characteristic required of metastatic cells is the ability toadapt to and survive the insults of pathophysiological stress before,during and after dissemination. Successful metastasis most likelyrequires the de novo expression, or activation of genes that augmentsurvival of tumor cells during periods of pathophysiological stress suchas hypoxia, loss of exogenous to growth factors, oxidative stress,immune response. Consequently, much research has been focused oncharacterizing genes involved in mediating the adaptive responses oftumor cells during periods of stress such as hypoxia resulting from alocal decrease in blood supply. Similar studies have also associated theability of tumor cells to survive and proliferate under hypoxicconditions with poor prognosis and resistance to radiation therapy. Itis not surprising then that the molecular mechanisms associated withtumor cell survival during hypoxic conditions are now being targeted fornovel therapeutic agents. Thus we feel it is important to identify andcharacterize clinically relevant metastatic gene markers induced inresponse to physiological stress.

Since breast cancer cells are particularly well known to adapt andsurvive periods of stress such as hypoxia (Knowles, et al., 2001.Hypoxia and Oxidative Stress in Breast Cancer: Hypoxia andtumourigenisis. Br Can Res 3, 318-322; Pugh, et al., 2001. Hypoxia andOxidative Stress in Breast Cancer: Hypoxia Signalling Pathways. Br CanRes 3, 313-317) accompanied by serum deprivation, we developed a methodto monitor the expression of a panel of genes identified as breast CTCidentification markers, during exposure of breast cancer cell lines tohypoxia, serum deprivation and a combination thereof. Our inventionreveals that serum deprivation alone, and especially serum deprivationin combination with hypoxia, lead to a dramatic increase in humananterior gradient-2 (Hag-2, AGR2) and intestinal trefoil factor-3 (TFF3,ITF3) mRNA expression. This invention provides a method and means intohow CTC markers are regulated in vivo and could ultimately aid in thedesign of novel therapies targeted at blocking breast cancer metastasis.

SUMMARY OF THE INVENTION

The present invention provides a method and means for diagnosing cancerby utilizing the role of AGR2 and TFF3 metabolism to physiologicalstress. Tumors from breast cancer patients express higher levels ofthese genes when compared to normal tissue when subjected to stress.After normalization to ubiquitin, AGR2 and TFF3 expression increases(approximately 60%) of patient matched tumor samples when compared tonormal tissue. This increase provides the foundation for assessingdisease state, response to therapy and other prognostic values. Themethod is applicable in cancers with overexpression of one or both genessuch as ovarian, lung and thyroid tumors, colon, stomach, rectum andprostate in an immunomagnetically enriched sample. Accordingly, breastcancer cells co-adapt the use of AGR2 and TFF3 to mediate cell survivaland repair, similar to their role in normal intestinal epithelial cells.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the expression levels after induction to stress. Panel Aand B depict the induction of AGR2 and TFF3 expression, respectively. Cainduction of VEGF is shown in panel C. Induction of S100A16 is shown inpanel D.

FIG. 2 Serum deprivation of MDA-MB-231 cells are shown. Panel A showsAGR2 induction blocked with ERK1/2 after both serum deprivation andhypoxia. Panel B shows inhibition of TFF3 was only inhibited after serumdeprivation. Panel C and D show a lack of inhibition with VEGF inductionand hypoxia.

FIG. 3 expression levels of AGR2 and TFF3 after normalization toubiquitin. A standard cancer blot was used to assess expression levelsin multiple tumor samples. Each sample shows an upregulation of thetumor sample compared with their mateched healthy tissue.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

The ability of tumor cells to metastasize to distant organs isresponsible for most cancer deaths. Despite a growing amount ofresearch, the molecular mechanisms associated with tumor progressiontowards metastasis remain poorly understood. In recent years, much ofthe research on this subject has been initiated on genes identified bytechniques such as microarray analyses and proteomic profiling of tumortissues and cell lines. These types of studies are, and will continue tobe, crucial to gaining insight into the multi-step processes of tumorprogression. Anterior gradient 2 (AGR2) is a recently to discoveredhuman homologue of the secreted Xenopus laevis protein XAG-2. XAG-2 isexpressed in the cement gland of Xenopus laevis and is associated withanteroposterior fate determination during early development. Sequenceanalysis of AGR2 revealed a predicted N-terminal cleavable secretorysignal that suggests it is a secreted protein in humans as well. Anincreased interest in AGR2 was derived from the original finding that itis co-expressed with the estrogen receptor in breast cancer cell lines.AGR2 expression in an enriched sample of circulating tumor cells derivedfrom breast, prostate and colon cancer patients would provide adiagnostic/prognostic tool in assessing these disease states. Thusmaking AGR2 a clinically relevant marker in cancer progression.

Induction of AGR2 and TFF3 During Stress Treatment.

Breast cancer lines are subjected to serum starvation, hypoxia and acombination thereof. These treatments mimic conditions experienced bybreast tumors during periods of pathophysiological stress resulting fromdecreased blood supply. Expression levels of genes, previouslyidentified to be potential candidates for breast cancer CTC detectionare measured by quantitative RT-PCR before and after stress inductionfor comparison. Serum deprivation alone, and especially serumdeprivation in combination with hypoxia, leads to a dramatic increase inthe expression of anterior gradient-2 (AGR2) and trefoil factor 3 (TFF3,ITF3) in breast cancer cells. The most dramatic induction of AGR2 andTFF3 expression is observed in the MDA-MB-231 cell line (FIGS. 1A andB). Co-induction of the classic hypoxia responding gene VEGF suggeststhat the hypoxia response pathway is activated during treatment of cells(FIG. 1C). In addition to these genes, the expression of S100A16 ismonitored to verify the observed induction of AGR2 and TFF3 which is aspecific response to the applied stress conditions (FIG. 1D). Afterrepeating stress treatment of MDA-MB-231 cells, similar levels ofinduction for HAG-2 and TFF3 are observed. These results, along withprevalent expression of HAG-2 and TFF3 in CTCs, suggest that AGR2 andTFF3 play an important role in a cellular response to hostile growthconditions experienced by tumor cells before and after dissemination.Thus, there is a suggestion that both AGR2 and TFF3 play a role inbreast cancer cell survival during periods of physiological stress.

Er1/2 Pathway is Involved in the Activation of AGR2 and TFF3Transcription During Stress Treatment.

Chemical inhibitors of ERK1/2, JNK, p38 and PI3K were used in an attemptto better understand the signaling pathways responsible for induction isof AGR2 and TFF3 during serum deprivation and hypoxic treatment ofMDA-MB-231 cells. Cells were treated with serum deprivation, hypoxia anda combination thereof for 48 hours in the presence and absence of eachinhibitor. After treatment and quantitative PCR analysis, we observedthat the ERK1/2 inhibitor, PD98059, was sufficient to block induction ofAGR2 by serum deprivation alone and its combination with hypoxia (FIG. 2A). In contrast, TFF3 induction was blocked by PD98059 only duringtreatment with serum deprivation. Interestingly, induction of TFF3 wasnot inhibited when serum deprivation was combined with hypoxia (FIG. 2B), suggesting alternative-signaling mechanisms are responsible foractivation of this gene in response to different stimuli. VEGF inductionand S100A16 expression were also not affected by treatment withinhibitors, further suggesting that the effect of PD98059 is specificfor blocking AGR2 induction (FIGS. 2 C and D).

Overexpression of AGR2 and TFF3 in Breast Tumors.

Because AGR2 and TFF3 play a significant role in the response of breastcancer cells to physiological stress, it to be advantageous for breasttumors to express higher levels of these genes when compared to normaltissue. Using a commercially available cancer-profiling array to comparethe expression of AGR2 and TFF3 in patient matched normal and breastcancer samples, AGR2 and TFF3 expression increases in approximately 60%of patient matched tumor samples when compared to normal tissue andafter normalization to ubiquitin (FIG. 3). The over-expression in breasttumors suggests a role in progression towards metastasis. Thus, duringphysiological stress, breast cancer cells co-adapt the use of AGR2 andTFF3 to mediate cell survival and repair, similar to their role innormal intestinal epithelial cells.

The present invention combines immunomagnetic enrichment of patientsamples as discussed in U.S. Pat. No. 6,365,362 and U.S. Pat. No.6,645,731 (both incorporated by reference) with a stress-inducedinduction of AGR2 and TFF3 to provide a method in cancer diagnosis.

1. A method for detecting and enumerating circulating tumor cells in amixed cell population, the presence of said cells in said populationbeing indicative of a disease state, comprising: a) preparing animmunomagnetic sample wherein a biological specimen from a test subject,which specimen comprises a mixed cell population suspected of containingsaid CTC cells, which CTC cells are present at 1 to 50 cells per ml, ismixed with magnetic particles coupled to a biospecific ligand whichreacts specifically with the CTC cells, to the substantial exclusion ofother sample components; b) inducing a physiological stress on the CTCcells; c) determining the expression levels of a gene from a groupconsisting of AGR2, TFF3, and combinations thereof; d) analyzing saidgenes and CTC cells wherein the greater the number of CTC cellsexpressing high levels of said gene the greater the severity of saiddisease state.
 2. A method as claimed in claim 1, wherein as anintermediate step between the preparation of the immunomagnetic sampleand inducing a physiological stress, said immunomagnetic sample issubjected to a magnetic field to produce an enriched CTC cell suspensionas the immunomagnetic sample.
 3. A method as claimed in claim 1, whereinsaid disease state is cancer.
 4. A method as claimed in claim 1, whereinsaid biospecific ligand is a monoclonal antibody specific for at leastone cancer cell determinant.
 5. A method as claimed in claim 1, whereinsaid method further comprises the step of assessing the malignant statusof the labeled cancer cell-containing fraction by immunocytochemicalanalysis.
 6. A method as claimed in claim 1, wherein said biologicalspecimen is obtained from said test subject periodically and assessedfor the presence and number of circulating cancer cells as an indicatorof either progression of said disease state, or the patient's responseto cancer eradication procedures.
 7. A method as claimed in claim 1,wherein said biospecific ligand binds specifically to an epithelial celladhesion molecule.
 8. A method as claimed in any of claims 1, whereinsaid biological specimen is peripheral blood
 9. The method according toclaim 3 wherein the cancer or carcinoma is selected from the groupconsisting of prostate cancer, breast cancer, colon cancer apudoma,choristoma, branchioma, malignant carcinoid syndrome, carcinoid heartdisease, carcinoma e.g., Walker, basal cell, basosquamous, Brown-Pearce,ductal, Ehrlich tumor, in situ, Krebs 2, merkel cell, mucinous,non-small cell lung, oat cell, papillary, scirrhous, bronchiolar,bronchogenic, squamous cell and transitional cell reticuloendotheliosis,melanoma, chondroblastoma, chondroma, chondrosarcoma, fibroma,fibrosarcoma, giant cell tumors, histiocytoma, lipoma, liposarcoma,mesothelioma, myxoma, myxosarcoma, osteoma, osteosarcoma, Ewing'ssarcoma, synovioma, adenofibroma, adenolymphoma, carcinosarcoma,chordoma, mesenchymoma, mesonephroma, myosarcoma, ameloblastoma,cementoma, odontoma, teratoma, throphoblastic tumor, adenocarcinoma,adenoma, cholangioma, cholesteatoma, cylindroma, cystadenocarcinoma,cystadenoma, granulosa cell tumor, gynandroblastoma, hepatoma,hidradenoma, islet cell tumor, leydig cell tumor, papilloma, sertolicell tumor, theca cell tumor, leiomyoma, leiomyosarcoma, myoblastoma,myoma, myosarcoma, rhabdomyoma, rhabdomyosarcoma, ependymoma,ganglioneuroma, glioma, medulloblastoma, meningioma, neurilemmoma,neuroblastoma, neuroepithelioma, neurofibroma, neuroma, paraganglioma,paraganglioma nonchromaffin, antiokeratoma, angioma sclerosing,angiomatosis, glomangioma, hemangioendothelioma, hemangioma,hemangiopericytoma, hemangiosarcoma, lymphangioma, lymphangiomyoma,lymphangiosarcoma, pinealoma, carcinosarcoma, chondrosarcoma,cystosarcoma phyllodes, fibrosarcoma, hemangiosarcoma, leiomyosarcoma,leukosarcoma, liposarcoma, lymphangiosarcoma, myosarcoma, myxosarcoma,ovarian carcinoma, rhabdomyosarcoma, sarcoma (Kaposi's, and mast-cell),neoplasms (e.g., bone, digestive system, liver, pancreatic, pituitary,testicular, orbital, head and neck, central nervous system, acoustic,pelvic, respiratory tract, and urogenital), neurofibromatosis, andcervical dysplasia.
 10. A method as claimed in claim 9 wherein thecarcinoma is a breast carcinoma.